Secretion of functional interferon by the type 3 secretion system of enteropathogenic Escherichia coli.

BACKGROUND: Type I interferons (IFN-I)-a group of cytokines with immunomodulatory, antiproliferative, and antiviral properties-are widely used as therapeutics for various cancers and viral diseases. Since IFNs are proteins, they are highly susceptible to degradation by proteases and by hydrolysis in the strong acid environment of the stomach, and they are therefore administered parenterally. In this study, we examined whether the intestinal bacterium, enteropathogenic Escherichia coli (EPEC), can be exploited for oral delivery of IFN-Is. EPEC survives the harsh conditions of the stomach and, upon reaching the small intestine, expresses a type III secretion system (T3SS) that is used to translocate effector proteins across the bacterial envelope into the eukaryotic host cells. RESULTS: In this study, we developed an attenuated EPEC strain that cannot colonize the host but can secrete functional human IFNα2 variant through the T3SS. We found that this bacteria-secreted IFN exhibited antiproliferative and antiviral activities similar to commercially available IFN. CONCLUSION: These findings present a potential novel approach for the oral delivery of IFN via secreting bacteria.

Predicting the pathogenicity of bacterial genomes using widely spread protein families.

BACKGROUND: The human body is inhabited by a diverse community of commensal non-pathogenic bacteria, many of which are essential for our health. By contrast, pathogenic bacteria have the ability to invade their hosts and cause a disease. Characterizing the differences between pathogenic and commensal non-pathogenic bacteria is important for the detection of emerging pathogens and for the development of new treatments. Previous methods for classification of bacteria as pathogenic or non-pathogenic used either raw genomic reads or protein families as features. Using protein families instead of reads provided a better interpretability of the resulting model. However, the accuracy of protein-families-based classifiers can still be improved. RESULTS: We developed a wide scope pathogenicity classifier (WSPC), a new protein-content-based machine-learning classification model. We trained WSPC on a newly curated dataset of 641 bacterial genomes, where each genome belongs to a different species. A comparative analysis we conducted shows that WSPC outperforms existing models on two benchmark test sets. We observed that the most discriminative protein-family features in WSPC are widely spread among bacterial species. These features correspond to proteins that are involved in the ability of bacteria to survive and replicate during an infection, rather than proteins that are directly involved in damaging or invading the host.

Monoclonal Antibody-Based Biosensor for Point-of-Care Detection of Type III Secretion System Expressing Pathogens.

It is predicted that the antibiotic resistance crisis will result in an annual death rate of 10 million people by the year 2050. To grapple with the challenges of the impending crisis, there is an urgent need for novel and rapid diagnostic tools. In this study, we developed a novel monoclonal antibody-named mAb-EspB-B7-that targets the EspB protein, a component within the bacterial type 3 secretion system (T3SS), which is mainly expressed in Gram-negative pathogens and is essential for bacterial infectivity. We found that mAb-EspB-B7 has high affinity and specificity toward recombinant and native EspB proteins; is stable over a range of pH levels, temperatures, and salt concentrations; and retains its functionality in human serum. We identified the epitope for mAb-EspB-B7 and validated it by competitive enzyme-linked immunosorbent assay (ELISA). Since this epitope is conserved across several T3SS-harboring pathogens, mAb-EspB-B7 holds great potential for development as an active component in precise and rapid diagnostic tools that can differentiate between commensal and pathogenic bacterial strains. To this end, we integrated the well-characterized monoclonal antibody into an electrochemical biosensor and demonstrated its high specificity and sensitivity capabilities in detecting pathogenic bacterial T3SS-associated antigens as well as intact bacteria. We foresee that in the near future it will be possible to design and develop a point-of-care biosensor with multiplexing capabilities for the detection of a panel of pathogenic bacteria.

BacPaCS-Bacterial Pathogenicity Classification via Sparse-SVM.

MOTIVATION: Bacterial infections are a major cause of illness worldwide. However, most bacterial strains pose no threat to human health and may even be beneficial. Thus, developing powerful diagnostic bioinformatic tools that differentiate pathogenic from commensal bacteria are critical for effective treatment of bacterial infections. RESULTS: We propose a machine-learning approach for classifying human-hosted bacteria as pathogenic or non-pathogenic based on their genome-derived proteomes. Our approach is based on sparse Support Vector Machines (SVM), which autonomously selects a small set of genes that are related to bacterial pathogenicity. We implement our approach as a tool-'Bacterial Pathogenicity Classification via sparse-SVM' (BacPaCS)-which is fully automated and handles datasets significantly larger than those previously used. BacPaCS shows high accuracy in distinguishing pathogenic from non-pathogenic bacteria, in a clinically relevant dataset, comprising only human-hosted bacteria. Among the genes that received the highest positive weight in the resulting classifier, we found genes that are known to be related to bacterial pathogenicity, in addition to novel candidates, whose involvement in bacterial virulence was never reported. AVAILABILITY AND IMPLEMENTATION: The code and the resulting model are available at: https://github.com/barashe/bacpacs. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The role of EscD in supporting EscC polymerization in the type III secretion system of enteropathogenic Escherichia coli.

The type III secretion system (T3SS) is a multi-protein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. In enteropathogenic Escherichia coli, a prevalent cause of diarrheal diseases, the needle complex base of the T3SS is formed by multi-rings: two concentric inner-membrane rings made by the two oligomerizing proteins (EscD and EscJ), and an outer ring made of a single oligomerizing protein (EscC). Although the oligomerization activity of these proteins is critical for their function and can, therefore, affect the virulence of the pathogen, the mechanisms underlying the oligomerization of these proteins have yet to be identified. In this study, we report that the proteins forming the inner-membrane T3SS rings, EscJ and EscD proteins, are crucial for the oligomerization of EscC. Moreover, we elucidate the oligomerization process of EscD and determine the contribution of individual regions of the protein to its self-oligomerization activity. We show that the oligomerization motif of EscD is located at its N-terminal portion and that its transmembrane domain can self-oligomerize, thus contributing to the self-oligomerization of the full-length EscD.

Deciphering the Mechanical Properties of Type III Secretion System EspA Protein by Single Molecule Force Spectroscopy.

Bacterial pathogens inject virulence factors into host cells during bacterial infections using type III secretion systems. In enteropathogenic Escherichia coli, this system contains an external filament, formed by a self-oligomerizing protein called E. coli secreted protein A (EspA). The EspA filament penetrates the thick viscous mucus layer to facilitate the attachment of the bacteria to the gut-epithelium. To do that, the EspA filament requires noteworthy mechanical endurance considering the mechanical shear stresses found within the intestinal tract. To date, the mechanical properties of the EspA filament and the structural and biophysical knowledge of monomeric EspA are very limited, mostly due to the strong tendency of the protein to self-oligomerize. To overcome this limitation, we employed a single molecule force spectroscopy (SMFS) technique and studied the mechanical properties of EspA. Force extension dynamic of (I91)4-EspA-(I91)4 chimera revealed two structural unfolding events occurring at low forces during EspA unfolding, thus indicating no unique mechanical stability of the monomeric protein. SMFS examination of purified monomeric EspA protein, treated by a gradually refolding protocol, exhibited similar mechanical properties as the EspA protein within the (I91)4-EspA-(I91)4 chimera. Overall, our results suggest that the mechanical integrity of the EspA filament likely originates from the interactions between EspA monomers and not from the strength of an individual monomer.

Proline localized to the interaction interface can mediate self-association of transmembrane domains.

Assembly of transmembrane domains (TMDs) is a critical step in the function of membrane proteins. In recent years, the role of specific amino acids in TMD-TMD interactions has been better characterized, with more emphasis on polar and aromatic residues. Despite the high abundance of proline residues in TMDs, contribution of proline to TMD-TMD association has not been intensively studied. Here, we evaluated statistically the frequency of appearance, and experimentally the contribution of proline, compared to other hydrophobic amino acids (Gly, Ala, Val, Leu, Ile, and Met), with regard to TMD-TMD self-assembly. Our model system is the assembly motif ((22)QxxS(25)) found previously in TMDs of the Escherichia coli aspartate receptor (Tar-1). Statistically, our data revealed that all different motifs, except PxxS (P/S), have frequencies similar to their theoretical random expectancy within a database of 41916 sequences of TMDs, while PxxS motif is underrepresented. Experimentally, using the ToxR assembly system, the SDS-gel running pattern of biotin-conjugated TMD peptides, and FRET experiments between fluorescence-labeled peptides, we found that only the P/S motif preserves the dimerization ability of wild-type Tar-1 TMD. Although proline is known as a helix breaker in solution, Circular Dichroism spectroscopy revealed that the secondary structure of the P/S and the wild-type peptides are similar. All together, these data suggest that proline can stabilize TM self-assembly when localized to the interaction interface of a transmembrane oligomer. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

The sequence of events of enteropathogenic E. coli's type III secretion system translocon assembly.

Many bacterial pathogens employ the type III secretion system (T3SS), a specialized complex that transports effector proteins that manipulate various cellular processes. The T3SS forms a translocon pore within the host-cell membrane consisting of two secreted proteins that transition from a soluble state into a transmembrane complex. Still, the exact sequence of events leading to the formation of a membranous functional pore remains uncertain. Here, we utilized the translocon proteins of enteropathogenic E. coli (EPEC) to investigate the sequence of those steps leading to translocon assembly, including self-oligomerization, hetero-oligomerization, interprotein interaction, and membrane insertion. We found that in EPEC, EspD (SctE) plays a dominant role in pore formation as it assembles into an oligomeric state, regardless of pH, membrane contact, or the presence of EspB (SctB). Subsequently, EspB subunits integrate into EspD homo-oligomers to create EspB-EspD hetero-oligomers that adopt a transmembrane orientation to create a functional pore complex.

A dual-channel electrochemical biosensor enables concurrent detection of pathogens and antibiotic resistance.

Diarrheagenic E. coli infections, commonly treated with ß-lactam antibiotics, contribute to antibiotic resistance - a pressing public health concern. Rapid monitoring of pathogen antibiotic resistance is vital to combat antimicrobial spread. Current bacterial diagnosis methods identify pathogens or determine antibiotic resistance separately, necessitating multiple assays. There is an urgent need for tools that simultaneously identify infectious agents and their antibiotic resistance at the point of care (POC). We developed an integrated electrochemical chip-based biosensor for detecting enteropathogenic E. coli (EPEC), a major neonatal diarrheal pathogen, using an antibody against a virulence marker, termed EspB, and the ß-lactam resistance marker, ß-lactamase. A dual-channel microfabricated chip, bio-functionalized with a specific EspB monoclonal antibody, and nitrocefin, a ß -lactamase substrate was utilized. The chip facilitated electrochemical impedance spectroscopy (EIS)-based detection of EspB antigen and EspB-expressing bacteria. For ß-lactam resistance profiling, a second channel enabled differential-pulse voltammetric (DPV) measurement of hydrolyzed nitrocefin. EIS-based detection of EspB antigen was calibrated (LOD: 4.3 ng/mL ±1 and LOQ: 13.0 ng/mL ±3) as well as DPV-based detection of the antibiotic resistance marker, ß-lactamase (LOD: 3.6 ng/mL ±1.65 and LOQ: 10 ng/mL ±4). The integrated EIS and DPV biosensor was employed for the simultaneous detection of EspB-expressing and ß-lactamase-producing bacteria. The combined readout from both channels allowed the distinction between antibiotic-resistant and -sensitive pathogenic bacteria. The integrated electrochemical biosensor successfully achieved simultaneous, rapid detection of double positive EspB- and ß-lactamase-expressing bacteria. Such distinction enabled by a portable device within a short assay time and a simplified sample preparation, may be highly valuable in mitigating the spread of AMR. This new diagnostic tool holds promise for the development of POC devices in clinical diagnosis.

EscE and EscG are cochaperones for the type III needle protein EscF of enteropathogenic Escherichia coli.

Type III secretion systems (T3SSs) are central virulence mechanisms used by a variety of Gram-negative bacteria to inject effector proteins into host cells. The needle polymer is an essential part of the T3SS that provides the effector proteins a continuous channel into the host cytoplasm. It has been shown for a few T3SSs that two chaperones stabilize the needle protein within the bacterial cytosol to prevent its premature polymerization. In this study, we characterized the chaperones of the enteropathogenic Escherichia coli (EPEC) needle protein EscF. We found that Orf2 and Orf29, two poorly characterized proteins encoded within the EPEC locus of enterocyte effacement (LEE), function as the needle protein cochaperones. Our finding demonstrated that both Orf2 and Orf29 are essential for type III secretion (T3S). In addition, we found that Orf2 and Orf29 associate with the bacterial membrane and form a complex with EscF. Orf2 and Orf29 were also shown to disrupt the polymerization of EscF in vitro. Prediction of the tertiary structures of Orf2 and Orf29 showed high structural homology to chaperones of other T3SS needle proteins. Overall, our data suggest that Orf2 and Orf29 function as the chaperones of the needle protein, and therefore, they have been renamed EscE and EscG.

Transmembrane domains interactions within the membrane milieu: principles, advances and challenges.

Protein-protein interactions within the membrane are involved in many vital cellular processes. Consequently, deficient oligomerization is associated with known diseases. The interactions can be partially or fully mediated by transmembrane domains (TMD). However, in contrast to soluble regions, our knowledge of the factors that control oligomerization and recognition between the membrane-embedded domains is very limited. Due to the unique chemical and physical properties of the membrane environment, rules that apply to interactions between soluble segments are not necessarily valid within the membrane. This review summarizes our knowledge on the sequences mediating TMD-TMD interactions which include conserved motifs such as the GxxxG, QxxS, glycine and leucine zippers, and others. The review discusses the specific role of polar, charged and aromatic amino acids in the interface of the interacting TMD helices. Strategies to determine the strength, dynamics and specificities of these interactions by experimental (ToxR, TOXCAT, GALLEX and FRET) or various computational approaches (molecular dynamic simulation and bioinformatics) are summarized. Importantly, the contribution of the membrane environment to the TMD-TMD interaction is also presented. Studies utilizing exogenously added TMD peptides have been shown to influence in vivo the dimerization of intact membrane proteins involved in various diseases. The chirality independent TMD-TMD interactions allows for the design of novel short d- and l-amino acids containing TMD peptides with advanced properties. Overall these studies shed light on the role of specific amino acids in mediating the assembly of the TMDs within the membrane environment and their contribution to protein function. This article is part of a Special Issue entitled: Protein Folding in Membranes.

EscI: a crucial component of the type III secretion system forms the inner rod structure in enteropathogenic Escherichia coli.

The T3SS (type III secretion system) is a multi-protein complex that plays a central role in the virulence of many gram-negative bacterial pathogens. This apparatus spans both bacterial membranes and transports virulence factors from the bacterial cytoplasm into eukaryotic host cells. The T3SS exports substrates in a hierarchical and temporal manner. The first secreted substrates are the rod/needle proteins which are incorporated into the T3SS apparatus and are required for the secretion of later substrates, the translocators and effectors. In the present study, we provide evidence that rOrf8/EscI, a poorly characterized locus of enterocyte effacement-encoded protein, functions as the inner rod protein of the T3SS of EPEC (enteropathogenic Escherichia coli). We demonstrate that EscI is essential for type III secretion and is also secreted as an early substrate of the T3SS. We found that EscI interacts with EscU, the integral membrane protein that is linked to substrate specificity switching, implicating EscI in the substrate-switching event. Furthermore, we showed that EscI self-associates and interacts with the outer membrane secretin EscC, further supporting its function as an inner rod protein. Overall, the results of the present study suggest that EscI is the YscI/PrgJ/MxiI homologue in the T3SS of attaching and effacing pathogens.

The transmembrane domains of the type III secretion system effector Tir are involved in its secretion and cellular activities.

Introduction: Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen and one of the major causes of gastrointestinal illness in developing countries. EPEC, similar to many other Gram-negative bacterial pathogens, possesses essential virulence machinery called the type III secretion system (T3SS) that enables the injection of effector proteins from the bacteria into the host cytoplasm. Of these, the translocated intimin receptor (Tir) is the first effector to be injected, and its activity is essential for the formation of attaching and effacing lesions, the hallmark of EPEC colonization. Tir belongs to a unique group of transmembrane domain (TMD)-containing secreted proteins, which have two conflicting destination indications, one for bacterial membrane integration and another for protein secretion. In this study, we examined whether TMDs participate in the secretion, translocation, and function of Tir in host cells. Methods: We created Tir TMD variants with the original or alternative TMD sequence. Results: We found that the C-terminal TMD of Tir (TMD2) is critical for the ability of Tir to escape integration into the bacterial membrane. However, the TMD sequence was not by itself sufficient and its effect was context-dependent. Moreover, the N-terminal TMD of Tir (TMD1) was important for the postsecretion function of Tir at the host cell. Discussion: Taken together, our study further supports the hypothesis that the TMD sequences of translocated proteins encode information crucial for protein secretion and their postsecretion function.

A single filament biomechanical study of the enteropathogenic Escherichia coli Type III secretion system reveals a high elastic aspect ratio.

Type III secretion systems (T3SSs) are syringe-like protein complexes used by some of the most harmful bacterial pathogens to infect host cells. While the T3SS filament, a long hollow conduit that bridges between bacteria and host cells, has been characterized structurally, very little is known about its physical properties. These filaments should endure shear and normal stresses imposed by the viscous mucosal flow during infection within the intestinal tract. We used atomic force microscopy (AFM) to probe the longitudinal and radial mechanical response of individual T3SS filaments by pulling on filaments extending directly from bacterial surfaces and later pressing into filaments that were detached from the bacteria. The measured longitudinal elastic moduli were higher by about two orders of magnitude than the radial elastic moduli. These proportions are commensurate with the role of the T3SS filament, which requires horizontal flexibility while maintaining its structural integrity to withstand intense stresses during infection.

EscA is a crucial component of the type III secretion system of enteropathogenic Escherichia coli.

The virulence of many Gram-negative pathogens is associated with type III secretion systems (T3SSs), which deliver virulence effector proteins into the cytoplasm of host cells. Components of enteropathogenic Escherichia coli (EPEC) T3SS are encoded within the locus of enterocyte effacement (LEE). While most LEE-encoded T3SS proteins in EPEC have assigned names and functions, a few of them remain poorly characterized. Here, we studied a small LEE-encoded protein, Orf15, that shows no homology to other T3SS/flagellar proteins and is only present in attaching and effacing pathogens, including enterohemorrhagic E. coli and Citrobacter rodentium. Our findings demonstrated that it is essential for type III secretion (T3S) and that it is localized to the periplasm and associated with the inner membrane. Membrane association was driven by the N-terminal 19 amino acid residues, which were also shown to be essential for T3S. Consistent with its localization, Orf15 was found to interact with the EPEC T3SS outer membrane ring component, EscC, which was previously shown to be embedded within the outer membrane and protruding into the periplasmic space. Interestingly, we found that the predicted coiled-coil structure of Orf15 is critical for the protein's function. Overall, our findings suggest that Orf15 is a structural protein that contributes to the structural integrity of the T3S complex, and therefore we propose to rename it EscA.

Assay for Type III Secretion in Escherichia coli.

The type III secretion system (T3SS) is crucial for the virulence of several pathogenic Escherichia coli species as well as for other gram-negative bacterial strains. Therefore, the ability to monitor this system constitutes a valuable tool for assessing the involvement of different proteins in bacterial virulence, for identifying critical domains and specific mutations, and for evaluating the antivirulence activities of various drugs. The major advantage of the T3SS secretion assay for E. coli over assays for other gram-negative pathogens is that it does not necessarily require specific antibodies. Here, we describe how to grow enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) strains under T3SS-inducing conditions, separate the supernatant fraction from the bacterial pellet, analyze this fraction on sodium dodecyl sulfate (SDS)-polyacrylamide gels, and evaluate the level of T3SS activity. We describe a qualitative analysis using Coomassie staining and a quantitative assay using western blotting.

Interactions and substrate selectivity within the SctRST complex of the type III secretion system of enteropathogenic Escherichia coli.

Many bacterial pathogens employ a protein complex, termed the type III secretion system (T3SS), to inject bacterial effectors into host cells. These effectors manipulate various cellular processes to promote bacterial growth and survival. The T3SS complex adopts a nano-syringe shape that is assembled across the bacterial membranes, with an extracellular needle extending toward the host cell membrane. The assembly of the T3SS is initiated by the association of three proteins, known as SctR, SctS, and SctT, which create an entry portal to the translocation channel within the bacterial inner membrane. Using the T3SS of enteropathogenic Escherichia coli, we investigated, by mutational and functional analyses, the role of two structural construction sites formed within the SctRST complex and revealed that they are mutation-resistant components that are likely to act as seals preventing leakage of ions and metabolites rather than as substrate gates. In addition, we identified two residues in the SctS protein, Pro23, and Lys54, that are critical for the proper activity of the T3SS. We propose that Pro23 is critical for the physical orientation of the SctS transmembrane domains that create the tip of the SctRST complex and for their positioning with regard to other T3SS substructures. Surprisingly, we found that SctS Lys54, which was previously suggested to mediate the SctS self-oligomerization, is critical for T3SS activity due to its essential role in SctS-SctT hetero-interactions.

Indole intercepts the communication between enteropathogenic E. coli and Vibrio cholerae.

Reported numbers of diarrheal samples exhibiting co-infections or multiple infections, with two or more infectious agents, are rising, likely due to advances in bacterial diagnostic techniques. Bacterial species detected in these samples include Vibrio cholerae (V. cholerae) and enteropathogenic Escherichia coli (EPEC), which infect the small intestine and are associated with high mortality rates. It has previously been reported that EPEC exhibit enhanced virulence in the presence of V. cholerae owing to their ability to sense and respond to elevated concentrations of cholera autoinducer 1 (CAI-1), which is the primary quorum-sensing (QS) molecule produced by V. cholerae. In this study, we examined this interspecies bacterial communication in the presence of indole, a major microbiome-derived metabolite found at high concentrations in the human gut. Interestingly, we discovered that although indole did not affect bacterial growth or CAI-1 production, it impaired the ability of EPEC to enhance its virulence activity in response to the presence of V. cholerae. Furthermore, the co-culture of EPEC and V. cholerae in the presence of B. thetaiotaomicron, an indole-producing commensal bacteria, ablated the enhancement of EPEC virulence. Together, these results suggest that microbiome compositions or diets that influence indole gut concentrations may differentially impact the virulence of pathogens and their ability to sense and respond to competing bacteria.

Quantitative proteomic analysis reveals formation of an EscL-EscQ-EscN type III complex in enteropathogenic Escherichia coli.

We characterized Orf5 and SepQ, two type III secretion (T3S) system proteins in enteropathogenic Escherichia coli, and showed that they are essential for T3S, associated with the bacterial membrane, and interact with EscN. Our findings suggest that Orf5 and SepQ are homologs of YscL and YscQ from Yersinia, respectively.